Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system

Hiroaki Mon, Jaeman Lee, Mai Fukushima, Yudai Nagata, Mie Fujii, Jian Xu, Oumi Nishi, Kazuhiro Iiyama, Takahiro Kusakabe

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca2+ and Sr2+ were able to replace Mg2+ and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.

Original languageEnglish
Pages (from-to)6813-6822
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume97
Issue number15
DOIs
Publication statusPublished - Aug 1 2013

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Apium graveolens
Bombyx
Baculoviridae
Endonucleases
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Mutation
Hemolymph
DNA
Metals
Ions
Research

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system. / Mon, Hiroaki; Lee, Jaeman; Fukushima, Mai; Nagata, Yudai; Fujii, Mie; Xu, Jian; Nishi, Oumi; Iiyama, Kazuhiro; Kusakabe, Takahiro.

In: Applied Microbiology and Biotechnology, Vol. 97, No. 15, 01.08.2013, p. 6813-6822.

Research output: Contribution to journalArticle

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AU - Mon, Hiroaki

AU - Lee, Jaeman

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AU - Nagata, Yudai

AU - Fujii, Mie

AU - Xu, Jian

AU - Nishi, Oumi

AU - Iiyama, Kazuhiro

AU - Kusakabe, Takahiro

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